Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme. The oxidative half-reaction catalyzed by the monooxygenase enzyme has been proposed to occur through the formation of a (hydro)peroxyflavin intermediate. In some two-component flavin-dependent systems the mechanism of flavin transfer involves protein-protein interactions between the flavin reductase and monooxygenase enzyme. Methods are presented that provide an alternative approach from flavin-bound monooxygenases to evaluate the kinetic properties and flavin transfer mechanism of the two-component flavin-dependent monooxygenase systems. © 2019 Elsevier Inc. All rights reserved.